The microsporidia are an extremely ancient and diverse group of obligate intracellular parasites. They have been shown to infect species from nearly all animal phyla including man. At least 5 genera of microsporidia have been reported to infect humans. As many as 80% of AIDS patients experience symptoms of chronic diarrhea. Two studies have shown a high rate of infection by the microsporidian Enterocytozoon bieneusi in patients with chronic diarrhea. Chronic diarrhea is both a symptom of AIDS and a cause of death in AIDS patients. Because of a lack of phylogenetic information, little is known about the relationships among the human microsporidian isolates and other species of microsporidia. The broad, long-term goal of this research is to develop a database of rDNA sequences from a wide variety of microsporidian species in order to identify the host reservoir for the microsporidia which infect humans. The specific aim of this project is to compare sequences from a broad range of microsporidian genera to those of microsporidian isolates from AIDS patients. Microsporidia from sources including food, pet and pest species, and from environmental sources such water will be included in the analysis. The small subunit rDNA will be amplified and sequenced directly by double stranded dideoxy sequencing. Universal primers, located in highly conserved regions of the rDNA, will be used to sequence the entire amplified region in both directions. In the event that the existing primers do not initiate primer extension, species specific primers will be synthesized. Comparative phylogenetic analysis will be accomplished by three methods: maximum parsimony (Swofford), maximum likelihood (Felsenstein) and a least squares transformation of a distance matrix (DeSote). Comparative phylogenetic analysis of the sequence data will be used to determine which microsporidia are closely related to the human isolates. This information will help determine the sources of these opportunistic human parasites. The phylogenetic information will also be useful to other researchers in designing diagnostic probes for the detection of human microsporidiosis, in determining drug susceptibility of microsporidia, and in the study of new human microsporidial isolates.